This project studies the regulation of expression of genes encoding lens fiber membrane proteins that may be involved in cell-cell communication. Particular emphasis is put on the study of major intrinsic protein (MIP). We have cloned the MIP gene and have begun to analyze the cis regulatory elements responsible for its lens-specific expression. A recombinant clone containing the human MIP gene was isolated from a human leukocyte genomic library using a bovine MIP cDNA (Gorin et al., Cell 39:49, 1984). This is the first lens-specific non-crystallin gene that has been cloned. Sequencing data indicates that this gene is 3.6 kbp long and contains four exons. The 5' flanking sequence of the gene contains a TATA box, two CCAAT boxes, and potential binding sites for the transcription factors Spl, NF-kB, and glucocorticoid receptor. Alu repeats are present in the 5' flanking region and third intron of the gene. In order to characterize the elements regulating expression of the MIP gene, 5' flanking DNA fragments of different lengths, synthesized by the polymerase chain reaction, were introduced upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. Transfection of these gene constructs into explanted embryonic chicken lens epithelia and analysis of transient gene expression indicated that 253 bp of 5' flanking sequence contains an active promoter. The human MIP gene promoter functions with approximately the same efficiency as the promoter for the mouse gamma2 crystallin gene (also expressed specifically in lens fibers). We are presently mapping the various cis regulatory elements responsible for the discrete temporal and tissue-specific expression of the MIP gene during lens fiber cell differentiation.